Melior Neurophysiology Models

1 Sept, 2011 MELIOR DISCOVERY, Inc.: Neurophysiology Models

Nerve Conduction / NeuropathyNeuromuscular Reflex Function

Spinal Reflex ExcitabilityCortical & Neuromuscular Evoked Potentials

Auditory Sensory Gating

Electrophysiology Models

1 Sept, 2011 MELIOR DISCOVERY, Inc.: Neurophysiology Models 2

NEUROPHYSIOLOGY ASSAY

Nerve Conduction


Chemo-neuropathy EvaluationPeripheral nerve amplitude and conduction velocity measurements

• Vincristine administered 2x / week (1.7 mg/kg sc) to mice for 10 weeks• Caudal (tail) nerve conduction velocity is increased by treatment

Bieri et al, 1997, J. Neurosci. Res. 50:821-8

75 µV

2 ms

1 Sept, 2011 MELIOR DISCOVERY, Inc.: Neurophysiology Models4

Conclusion: • Vehicle and veh/IGF treated animals showed a normal increase in

caudal tail CV over 10 wks of treatment• Vincristine (Vin/Veh) treatment caused a reduction in CV over this

time• The vincristine-induced decrease was ameliorated by IGF-I.

IGF-I Protects Against Vincristine Reduction in Conduction Velocity (CV)

Change in CV from pre-treatment baseline values


• Gait measures (ipsi- and contralateral limb support) were reduced by

vincristine treatment. IGF-I (1 mg/kg sc) reduced the effect of

vincristine.

• Hot plate latency was increased by vincristine treatment. The increase

was prevented by IGF-I (1 mg/kg sc).

• Axonal pathology (abnormal axons and myelin) produced by vincristine

treatment was prevented by IGF-I (1 mg/kg sc).

• Body weight was not affected by vincristine or IGF-I.

Behavioral and Morphological Protectionby IGF-I in Vincristine Chemoneuropathy


American Journal of Pathology, Vol. 155, No. 2, August 1999

Copyright © American Society for Investigative Pathology

Neurophysiological, Behavioral, and Morphological Evaluation of SOD-KO Mice

• Mice lacking cytoplasmic Cu/Zn superoxide dismutase (SOD) were used as a model of the neurodegenerative effects of familial ALS.

• Caudal (mixed, tail), sural (sensory), and tibial (motor) nerve conduction velocity and amplitudes were evaluated at 5 – 7 mos of age.

• Rod-running latency and stride length were evaluated at 4, 6, and 14 mos.• Nerve histology and muscle histochemistry (SDH; red vs white fibers)

were evaluated at 2 and 6 mos.


Conduction Velocity & Amplitude Changes

Tibial (motor)

distal

proximal

Sural (sensory)

Caudal (mixed)

Suralnerve

.05 ms10 mA


Nerve Conduction Velocities and Amplitudes at 5–7 Months of Age in SOD -/- Mice

Wild type KO

**

*

Conclusion: SOD KO mice showed significant reductions in the conduction velocity of the caudal (tail) and tibial nerves, and in the latency of the plantar muscle response to tibial nerve stimulation.


Nerve Conduction in Adult SD Rats

Sciatic notch

Ankle

50 µs10 mA

Tibial (motor) nerve recording

∆ x

Tibialnerve

Ave of10 sweeps ISI: 2 sec

Sciatic

100

0

-100

250

0

-250

Am

plitu

de (

µV

)

4.2 msec

6.8 msec

Latency difference: (6.8 – 4.2) msec = 2.6 msecDistance: 40 mmConduction Velocity: 40 mm / 2.6 msec = 15.4 m/sec

Tibial

0 20-10 10

Actual Data, Adult Rat



50 µs10 mA

Sural (sensory) nerve recording

Suralnerve

∆ x

Am

plitu

de (

µV

)

0.75 msec


Latency difference: 0.75 msec Distance: 23 mmConduction Velocity: 23 mm / 0.75 msec = 31 m/sec

50

0

-100

50

0 2 6-2 4


Stimulus artifact

response



Proximal

Distal

200

0

-200

250

0

-250

Am

plitu

de (

µV

)

0 10 20 30-103.0 msec

5.5 msec


Latency difference: (5.5 – 3.0) msec = 2.5 msecDistance: 50 mmConduction Velocity: 50 mm / 2.5 msec = 20 m/sec


50 µs10 mA

0

5

10

cm Proximal

Distal

Caudal (mixed) nerve recording


Spinal Reflex Excitability:

C-fiber and Monosynaptic Reflexes


“Early” responseAδ/Aβ fibers

“Late” response C-fibers

100 200 3000 400

Stimulus

Time (msec)

Plantarnerve

Peroneus l. muscle

Spinalcord

Peronealnerve

Hindfoot2 ms

Method for Recording PlantarAδ/Aβ and C-fiber Responses (CFR)

C-fibers are small unmyelinated fibers transmitting diffuse pain signalsAδ/Aβ fibers are larger myelinated fibers transmitting pain and touch information


“early”10 - 25 msec

Aδ/Aβ fiber response

“late”150 - 400 msecC-fiber response

Characterization of C-fiber Reflex (CFR)

• C-fiber response latency consistent with conduction in unyelinated C-fibers (0.5 - 1 m/sec) rather than myelinated Aδ/Aβ fibers (12-20 m/sec)

• Threshold of late response ~4x higher than early response

• Capsaicin causes desensitization of late response consistent w/ C-fibers

“C-fibers” are small unmyelinated axons mediating pain responses. They produce polysynaptic activation of spinal motoneurons and reflex muscle contractions – the “Late” response shown above.


Quantification of C-fiber reflex

Peroneal muscle EMG response

RectifiedResponse

375 msec150

∫375

t = 150

Vi = V(t) dt ∑10

i = 1

ViCFR = ( ) / 10

Average over 1 minIntegrate over 225 msec Time from start (min)

Am

plitu

de

(no

rmalized

%)

6 sec 2 msec x 10 mA

EMGStimulus

0

25

50

75

100

125

150

0 20 40 60

Integrated LHLIntegrated RHLIntegrated LHLIntegrated RHL

CFR Quantification


Peroneus l. muscle

Tibialis anterior

Biceps femoris (isolated)

Soleus

100 msec

Biceps (isolated)

100 msec

Determination of afferent

nerve pathway

Determination of muscle of origin

Peroneus l. muscle response

After transection of sural nerve

100 msec

After transection of plantar nerve

Verification of CFR Pathway

The C-fiber response is produced by signals traveling in the plantar n. and activating motoneurons of the Peroneus L. muscle


Capsaicin30 µl x 0.4 mg/mlat stimulation site

Effect of Capsaicin on C-fiber Response

18 s

24 s

30 s

36 s

42 s

48 s

54 s

6 s

12 s

-5 s

Capsaicin initially enhances (6 & 12 sec) and then blocks the late response, consistent with desensitization of vanilloid receptors on C-fiber terminals.


Increased response at 3 mg/kg presumed to result from supra-spinal disinhibition

relative to spinal inhibition

Per

cent

cha

nge

in r

espo

nse

Time relative to injection (min)

0

20

40

60

80

100

120

140

160

180

-25 -15 -5 0 5 15 25 35

10 mg/kg

5.5 mg/kg

3 mg/kg

PBS

Morphine administered scat time 0. N=3 rats per curve.

Effect of Morphine on CFR

Morphine produces a biphasic dose response effect on the C-fiber reflex, enhancing it at 3 mg/kg and suppressing it at higher doses.


Morphine-Induced Inhibition of CFR is Reversed by Naloxone

Average CFR’s from R & L hind limbs in 1 rat

0

50

100

150

200

250

300

350

400

-20 -10 0 10 20 30 40 50

CF

R a

mp

litu

de,

% b

asel

ine

Morphine10 mg/kg sc

Naloxone0.4 mg/kg sc

Time relative to first injection (min)

baseline

*

#


Determination of Site of Drug Action

• The earliest site of action would be at the P2X or related vanilloid receptors in the sensory nerve endings, which can be tested by binding or other in vitro studies.

• Action of the drug on the unmyelinated axons in the sensory nerve can be identified by recording the amplitude of the compound (plantar) nerve action potential compared with the CFR amplitude.

• The efferent pathway from the spinal cord to the plantar muscles can be tested by directly stimulating the peroneal nerve and recording the peroneus muscle (“M”) responses.

• A spinal site of action can be evaluated by recording the dorsal horn field potential, which reflects the ability of the C-fiber afferents entering the cord to activate dorsal horn interneurons.

• A supraspinal site of action can be evaluated by determining the effect of transection of the dorslal-lateral descending columns which modulate spinal excitability.

Having established a model for evaluating analgesic effects of drugs via changes in the C-fiber reflex, the site of drug action needs to be identified.


Peroneal muscle EMG

50 msec

Plantar nerve

0

20

40

60

80

100

0 3 6 9 12 15

Stimulus current (mA)

Inte

grat

ed E

MG

/ C

AP

(% m

ax.)

Peroneal m.EMG

Plantar n.APV

Stimulus-Response Recruitment

Peroneus l.muscle

Hind footstimulation

2 ms14 → 0 mA

Spinalcord

Plantar nerve afferent volley

Conduction velocity = 0.5 - 1.0 m/s

Integration window

Plantar n. Afferent Volley versus CFR


Test Agent Does Not Inhibit Plantar Nerve C-fiber Afferent Volley

Mean effect of test agentEffect of test agent vs. time

Per

cent

cha

nge

in r

espo

nse

CFR Plantar n. APV

0

20

40

60

80

100

120

Veh. Test agent Veh. Test agent

p=0.013

p>0.05

N=4 N=4

0

1000

2000

3000

-20 -10 0 10 20

Inte

grat

ed a

ctiv

ity

Peroneusl. muscle

EMG

Plantar n. volley4000

Time relative to injection (min.)

Test agent3 mg/kg i.v.

The C-fiber response but not the amplitude of the plantar n. volley is reduced by the test drug => the drug is not acting on the efferent pathway.


Effect of Test Agent on the Efferent Peroneal Neuromuscular Pathway

Plantarnerve

2 ms10 mA

Spinalcord

Peroneus l. muscle

EMG

Peronealnerve

.05 ms10 mA

0

100

200

300

400

500

600

-40 -20 0 20 40Per

on

eal

mu

scle

am

pli

tud

e

Hind foot stimulatedC-fiber response

(mv*msec)

Peroneal n. directM-response

(mV, 25x)

Time (min) post injection

-42

4

-26

24

2 msec

Time (min)relative totest agent

injection(3 mg/kg iv)

100 msec

M-response C-Fiber Response

The direct M response is not effected by the test drug => drug is not acting on the efferent path.


Chronic Dorsal-lateral Funiculus (DLF) Lesion and CFRT9 cord

DLF lesion

Test Agent3 mg/kg iv.

0

2000

4000

6000

8000

-20 -10 0 10 20 30 40

Time post injection (min)

Inte

gra

ted

EM

G a

ctiv

ity

CF

R A

mp

litu

de

(mv*

ms/

100)

0

40

80

120

160

Preinjection

15’ Postinjection

62.5%p<0.0001

N= 10

The test agent blocked the CFR in normal animals (not shown), and also blocked it in animals with chronic DLF lesions.

Chronic DLF lesions were made in rats ~4 weeks prior to evaluation of a test agent on the CFR. Spinal lesions did not block the response to morphine or naloxone (not shown).

Lesion of the DLF pathway does not block CFR inhibition produced by test agent => drug does not act at supraspinal level.


Peroneusl. muscle

100 msec

Plantarnerve

2 ms14 mA

L4 Spinalcord

Peroneus l.muscle EMG

myelinated afferent

response C-fiber DHEP:

DHEP amplitude:Hind foot

stimulation

10xgain

Peronealnerve

Spinal Cord Dorsal Horn Field Potentials Plus CFR Recording


Test Agent Does Not Inhibit Dorsal Horn Evoked Potential

Effect of test agent vs. time Mean response inhibitionby test agent

0

20

40

60

80

100

120

140

-10 0 10 20 30 40 50

% c

han

ge

in a

mp

litu

de

CF

R v

s. D

HE

P

C-fiber refles

Dorsal hornevoked potential

Time from injection (min)

Test agent3 mg/kg iv.

Per

cen

t in

hib

itio

n

80

60

40

20

0

CFRamplitude

DHEPamplitude

N=3

p< 0.05

N.S.

The test drug does not reduce the amplitude of the dorsal horn evoked potential => the drug does not impair transmission between primary efferent terminals and the first-order spinal interneurons in the dorsal horn.


Monosynaptic Spinal Reflex


Spinal Reflex Excitability:Spinal Monosynaptic (H-) Reflex

• The Hoffman or “H” reflex is the monosynaptic muscle reflex produced by activating proprioceptive muscle afferents; aka the common achilles tendon-tap reflex

• Stimulation of the tibial nerve activates axons innervating the plantar muscle, producing a direct “M” or muscle response, and also proprioceptive afferents traveling to the spinal cord, which then activate spinal motoneurons producing a second muscle response.

• Unlike the CFR, the H-reflex does not directly involve any excitatory or inhibitory interneurons. Thus drugs that affect e.g. GABA receptors or release should not affect this reflex unless (like GABA-A agonists) they tonically increase GABAergic tone, whereas they do impair the C-fiber reflex.

Proprioceptiveafferents

0.5 ms1-10 mA

Spinal cord

Hindfoot

Tibialnerve

Spinal interneurons

Motor neurons

Plantarmuscle

Muscle (“M”) response

Monosynaptic(“H”) response

DRG


Characterization of the Plantar H- (Monosynaptic) Reflex

-10 -5 0 5 10 15

Time (ms)

EMG(mV)

M-response H-reflex

stimulus

• Stimulation of the tibial nerve produces a direct muscle (M) response in the plantar muscle starting about 3 msec after the stimulation, followed by an H (monosynaptic) reflex response at about 10 msec.

• GABA-A receptor agonist drugs typically reduce this response, while antagonists facilitate it, assuming the drugs penetrate the blood-brain barrier. Benzodiazepines typically have no effect.

• A drug that directly affects peripheral axons or neuromuscular junctions (e.g. ssuccinylcholine) should inhibit this reflex.


H- or monosynaptic reflex (MSR) responses from rat at various times before and after injection of either vehicle or 0.5 mg/kg IV diazepam. Each waveform is the average of 10 successive responses obtained at 6 sec intervals. Red biphasic square wave at time 0 represents stimulus pulse. Scale at bottom right in mV applies to all recordings. Diazepam, a benzodiazepine, has no effect on monosynaptic reflexes.

10 min before Vehicle inject.

Time ofVehicle inject.

10 min beforeDrug inject.

Time of Drug

inject.

10 min afterDrug

inject.

20 min afterDrug inject.

30 min afterDrug inject.

Time (msec)-5 0 5 10 15

02.04.0

-4.0-2.0

-6.0

MS

R A

mp

litu

de

M response

H response

(Lack of) Effect of Diazepam on H-Response

0

200

400

600

800

1000

1200

1400

-20 0 20 40 60 80 100Time (min)

Pea

k-P

eak

Am

pli

tud

e (µ

V)

M response

Vehicle

H response

Diazepam0.5 mg/kg IV


Cortical and NeuromuscularEvoked Potentials


Magnetic Motor Stimulation: Basic Principles and Clinical Experience(EEG Suppl. 43; chapter 25, pps. 293-307

Assessment of Spinal Cord Function


SEP

ASR

Motorfunction

SomatosensoryEvoked Potentials

Auditory StimulatedResponses

Cerebellar MyoelectricEvoked Responses

Conclusion: Sensory and motor evoked potentials provide a reliable means of monitoring recovery after spinal injury.


Auditory Sensory Gating Responses

Effect of Amphetamine


Paradigm:

2. Electrodes implanted in rats under sodium pentobarbital anesthesia:

• Left frontal cortex - left sensory-motor cortex (above hippocampus)

• Depth electrode, right CA3 region of the HC, referenced to a skull screw• Neck EMG

3. One week after recovery, animal exposed to auditory tones as follows

• Pairs of 5 k Hz tones, 10 ms duration, 0.5 s apart

• 10 s interval between pairs of tones

4. Outcome:

• Amplitude = P1 - N1, mV (most robust effect)

• Outcome = ratio of amplitude of second (test) to first (conditioning) response.

Evaluation of Attention by Auditory Sensory Gating Response

Stereotaxically placedelectrodes 4.0 mmbelow dura in the hippocampal CA-3 region

skull


Effect of Amphetamine on Auditory Gating Responses

• Rats were chronically implanted with screw electrodes over frontal and sensory-motor cortices, and with a bipolar metal electrode into the CA3 region of the hippocampus (electrode tip separation ~ 1 mm).

- Test tones were applied during surgery to optimize electrode placemnt

• Post surgical recovery, animals were placed into recording chamber and exposed to paired tones:

- 3 k Hz, 10 ms duration- 0.5 s interval between test tones- 10 s between pairs of test tones

• Three sets of 30 stimulus tone pairs were delivered at ~ 6 min intervals while the rat was awake and resting

• Amphetamine (1 or 3 mg/kg ip) was then administered• 10’, 20’, and 30’ post drug administration, additional sets were

recorded.• Individual peak amplitudes were analyzed and compared as a

function of “Conditioning” vs “Test” tone pulses, and drug: “Pre” vs “Amphetamine”.


Typical Auditory Evoked Potentials

F011_EEG

-1.0

-0.5

0.0

0.5

1.0

1.5

0 0.05 0.1 0.15

EP

Am

p (m

V) Cond.

Test

N1

P1

N2

P0

Surface recording

F011_CA3

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

0 0.05 0.1 0.15

Cond.

Test

N1

P1

N2

P0

CA-3 Recording




• P0 not well defined in EEG records, but N1 and P1 were• P0, N1, and P1 were well defined in CA-3 records• In both surface and CA3 recordings, identified potentials occurred at

similar latencies in both Conditioning and Test responses• P0 and N1 were slightly delayed from EEG to CA3 (~ xx ms), while P1

and N1 showed similar latencies: Are the potentials analagous??

Analysis of Peak-Peak Amplitudes of Auditory Evoked Potentials

Mean latencies (N= 3 responses) for the (P1 - N1) amplitude difference as a function of (conditioning vs test) and (Pre drug vs Amphetamine).

P1-N1

0.0

0.5

1.0

1.5

2.0

2.5

Pre Drug Amphetamine1 mg/kg IP

Am

pli

tid

e (

mV

)

Cond.

Test

(Cond vs Test): ANOVA, p= 0.02

VCVT


Analysis of percent inhibition of the test tone for various amplitude measures

0

20

40

60

80

100

P0-N1 P1-N1 P0-N1+P1

% I

nh

ibit

ion

Pre drug

Amphet.

*

**

p= 0.023

p= 0.009

p= 0.008

unpaired t-test, N= 3

% Inhibition = (VC – VT) * 100

VC

100% = complete inhibition; 0% = no effect

Amphetamine reduced inhibition of the Test evoked potential by all measures, with P1-N1 and P0-N1+P1 showing the most robust effect.


Evoked Potential Peak-Peak Measure


Effect of Amphetamine on Gating ResponsesPre Drug

-0.8

-0.4

0.0

0.4

0.8

20 40 60 80 100 120140

Time (ms)

Am

pli

tud

e (m

V)

N1

P1 Conditioning

Test

Tone

Post Amphetamine1 mg/kg IP

-0.8

-0.4

0.0

0.4

0.8

20 40 60 80 100120 140

Time (ms)

N1

P1

0

20

40

60

80

100

1.0 3.0

Amphetamine (mg/kg ip)

Per

cen

t in

hib

itio

n

of

Tes

t R

esp

on

se

Pre dosing

Post dosing

p< 0.001

p= 0.001

N=9 N=5

N = # of rats; P1-N1 amplitudes

Conditioning

Test

Am

pli

tud

e (m

V)

Amphetamine at both 1 and 3 mg/kg IP reduced inhibition of the auditory evoked gating responses.


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